The purpose of this project is to contribute to the understanding of hormone action on metabolic processes in normal breast tissue and to understand the aberrant response to hormones in hyperplastic or neoplastic breast tissue. Modified proteins in mouse and human breast tissues and milk are investigated to determine changes that may occur during differentiation. Explants from mammary tissue of mice are incubated in a defined synthetic medium in the presence or absence of peptide and steroid hormones and labeled with appropriate radio-isotopes to identify phosphorylated and glycosylated proteins by autoradiography following electrophoresis on polyacrylamide gels. Prolactin induced the phosphorylation of caseins and a protein of approximately 60,000 molecular weight. The latter corresponded to a protein present in colostral but not in mature milk. To investigate changes in the modified proteins of human milk that may occur throughout lactation, proteins in colostrum and milk were separated by electrophoresis on polyacrylamide gels. Using the metachromatic cationic carbocyanine dye, Ethyl Stains-all (ESA), some phosphorus- and sialic acid-containing proteins were found in colostral but not mature milk. Proteins that migrated at the same molecular weight on SDS polyacrylamide gels could be distinguished from one another by two-dimensional electrophoresis and by distinctive staining with ESA. Many laboratories have experienced problems in identifying and characterizing the kappa casein of human milk. This protein was identified by analogy with bovine kappa casein with respect to its staining properties with ESA and its chymosin sensitivity.